The efficient synthesis of fluorescent and non-fluorescent multivalent neoglycoconjugates is described by means of the Cu(i) catalyzed azide-alkyne 1,3-dipolar cycloaddition ("click-chemistry"). A well-defined glycopolymer, glycocyclodextrin or glycocluster architecture displaying galactose or lactose epitopes has been chosen. Cellular assays using U-937 and RAW 264.7 monocyte/macrophage cells showed that these glycocompounds have the capability to act as synthetic activators mimicking the lipopolysaccharide (LPS) effects. Thus, the click compounds promote cell adhesion and stimulation of monocytes, measured as an increase in the amount of TNFalpha, facilitating their differentiation to macrophages.

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.

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BackgroundL‐Leu and its metabolite β‐hydroxy‐β‐methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L‐Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L‐Leu and HMB in myotubes grown in the absence of any catabolic stimuli.MethodsStudies were conducted in vitro using rat L6 myotubes under normal growth conditions (non‐involving L‐Leu‐deprived conditions). Protein synthesis and mechanistic target of rapamycin signalling pathway were determined.ResultsOnly HMB was able to increase protein synthesis through a mechanism that involves the phosphorylation of the mechanistic target of rapamycin as well as its downstream elements, pS6 kinase, 4E binding protein‐1, and eIF4E. HMB was significantly more effective than L‐Leu in promoting these effects through an activation of protein kinase B/Akt. Because the conversion of L‐Leu to HMB is limited in muscle, L6 cells were transfected with a plasmid that codes for α‐keto isocaproate dioxygenase, the key enzyme involved in the catabolic conversion of α‐keto isocaproate into HMB. In these transfected cells, L‐Leu was able to promote protein synthesis and mechanistic target of rapamycin regulated pathway activation equally to HMB. Additionally, these effects of leucine were reverted to a normal state by mesotrione, a specific inhibitor of α‐keto isocaproate dioxygenase.ConclusionOur results suggest that HMB is an active L‐Leu metabolite able to maximize protein synthesis in skeletal muscle under conditions, in which no amino acid deprivation occurred. It may be proposed that supplementation with HMB may be very useful to stimulate protein synthesis in wasting conditions associated with chronic diseases, such as cancer or chronic heart failure.

Gene delivery systems based on the beta-cyclodextrin scaffold have been synthesized by combining the copper(I)-catalyzed azide-alkyne coupling ("click chemistry") and an efficient acylation method of the secondary hydroxyls; molecular flexibility, charge density and hydrophobic-hydrophilic balance are critical parameters that can be fine-tuned by the click approach.

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

Aims/hypothesis The aim of this study was to investigate the action of the glucose-lowering compound sodium tungstate on glucose transport in muscle myotubes and to unravel the molecular events underlying the effects observed. Methods We studied the effects of tungstate on 2-deoxy-Dglucose uptake, levels and translocation of the glucose transporters GLUT4 and GLUT1, and Glut4 (also known as Slc2a4) promoter activity. We also measured the modifications of individual components of the signalling pathways involved in the effects observed. Results Tungstate increased 2-deoxy-D-glucose uptake in differentiated L6 myotubes through an increase in the total amount and translocation of GLUT4 transporter. The effects on glucose uptake were additive to those of insulin. Tungstate activated transcription of the Glut4 promoter, as shown by an increase in Glut4 mRNA, and by a promoter reporter assay. The assay of deletions of the Glut4 promoter indicated that the effect of tungstate is mediated by the myocyte enhancer factor 2 (MEF2)-binding domain. Accordingly, MEF2 levels and DNA binding activities were increased in response to the treatment. Tungstate-induced glucose uptake and GLUT4 transcriptional activation were dependent on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), while no changes were observed in the phosphorylation state of the β subunit of the insulin receptor, in the phosphatidylinositol 3-kinase pathway or in the activation of 5′AMP-activated protein kinase. Conclusions/interpretation Tungstate activates glucose uptake in myotubes through a novel ERK1/2-dependent mechanism. This effect is exerted by an increase in the content and translocation of the GLUT4 transporter. This is the first report of a glucose-lowering compound activating Glut4 transcription through an ERK1/2-dependent increase in MEF2 levels.

The aim of the present study was to investigate the maternal-fetal transport, incorporation, and effects on liver delta-6 fatty-acid desaturase activity of dietary trans fatty acids in pregnant rats. Three groups of six rats each were fed three experimental diets containing approximately 0%, 15%, and 30% of trans fatty acids but containing the same proportion of linoleic (18:2 n-6) and a-linolenic (18:3 n-3) acids for 10 wk. On d 20 of pregnancy, the animals from each group were killed. We determined the fatty acid profiles in plasma, brain, and liver microsomes of pregnant rats, as well as in placenta and fetal liver and brain. No changes were found in the number of fetuses of the pregnant rats. Trans fatty acids were incorporated in high concentrations in placenta and in maternal and fetal tissues, except brain, strongly elevating the linoleic acid proportion and lowering that of docosahexaenoic acid. The delta-6 fatty-acid desaturase activity in the liver microsomes of the pregnant rats was inhibited by trans isomers. In conclusion, high intakes of trans fatty acids partially inhibit liver delta-6 fatty-acid desaturase in pregnant rats, which may explain, in part, the low concentrations of docosahexaenoic acid in pregnant and fetal tissues. However, the fatty acid composition of both fetal and pregnant rat brain remains mostly unaffected regardless of the dietary trans fatty acid content.